We have found that chemotherapy and radiation converts the tumor site into an antigen-presenting cell-rich environment, rendering it ideal for the direct injection of a peptide or protein vaccine into the tumor to elicit potent antigen-specific T cell-mediated immune responses. Here, we propose the use of a therapeutic HPV protein vaccine, administered intratumorally, during chemoradiation in patients with inoperable HPV16+ cervical cancer. We have secured the therapeutic HPV protein vaccine TA-CIN from Cancer Research Technology, Ltd. and the adjuvant GPI-0100 from Hawaii Biotech. We have also secured support from the NCI NExT program to formulate and vial clinical grade TA-CIN with GPI-0100. The proposed trial will be performed at the University of Alabama at Birmingham. Specifically we plan to: 1) evaluate the safety and toxicity of TA-CIN with GPI-0100 administered intratumorally in inoperable HPV16+ cervical cancer patients; 2) characterize the HPV-16 E6 and E7 cell-mediated immune responses and L2-specific antibody titers in advanced cervical cancer patients intratumorally vaccinated with TA-CIN/GPI-0100; 3) determine the subset population of immune cells infiltrating the lesion bed, the expression of PD-L1 in the tumor microenvironment and the apoptotic tumor cell death in HPV 16+ advanced cervical cancer patients receiving intratumoral TA-CIN/ GPI-0100 vaccination; and 4) characterize the HPV-16 antigen-specific CD8+ T cell-mediated immune responses and therapeutic antitumor effects against E6/E7-expressing tumors in tumor-bearing mice treated with intratumoral vaccination with TA-CIN/GPI-0100, TA-HPV vaccinia or pNGVL4a-hCRTE6E7L2 DNA vaccine in conjunction with cisplatin and/or radiation treatment. The successful implementation of the proposed project will not only improve the treatment of HPV-associated advanced cervical cancer but may also serve as a platform technology for the development of effective therapeutic vaccines for other HPV associated malignancies including vaginal, vulva, anal, penile and HPV positive oropharyngeal squamous cell carcinoma.